1. Field of the Invention
The present invention relates to a laser microscope apparatus.
This application is based on Japanese Patent Application No. 2008-135523, the content of which is incorporated herein by reference.
2. Description of Related Art
There is a known coherent anti-Stokes Raman scattering microscope with which a specimen is observed through generation and detection of coherent anti-Stokes Raman scattering light from a molecule in the specimen utilizing its specific molecular vibration (for example, refer to Japanese Unexamined Patent Application, Publication No. 2002-520612). Since this coherent anti-Stokes Raman scattering microscope utilizes a specific molecular vibration in the specimen, there is no need to previously label the observation target with a fluorescence probe likewise of a fluorescence microscope. In addition, the molecule for observation can be changed by changing the vibration to use.
Conventionally, for the light source of such a coherent anti-Stokes Raman scattering microscope, a picosecond pulsed laser having two different frequencies of relatively narrow frequency spectral bands has been used. In a state where the frequency difference between these two picosecond pulsed laser beams is adjusted to correspond to the specific molecular vibration frequency in the specimen, light is focused on the specimen surface. At this time, in an extremely narrow space of a high photon density spreading in the vicinity of the focal plane, the frequency difference between these two picosecond pulsed laser beams resonates with the specific molecular vibration frequency to thereby generate strong coherent anti-Stokes Raman scattering light. This coherent anti-Stokes Raman scattering light has a higher frequency (that is to say, a shorter wavelength) than the frequencies of the irradiated two picosecond pulsed laser beams. Accordingly, the molecule in the specimen can be observed by spectrally selecting and detecting this coherent anti-Stokes Raman scattering light alone.
In addition, there is also known a multiphoton excitation type laser microscope in which femtosecond pulsed laser beams are focused on the specimen surface to thereby increase the photon density in an extremely narrow space spreading in the vicinity of the focal plane so as to cause multiphoton excitation of a fluorophore so that a sharp fluorescence image can be obtained (for example, refer to Japanese Unexamined Patent Application, Publication No. 2002-243641).
However, in order to efficiently generate coherent anti-Stokes Raman scattering light from a specific molecular vibration in the specimen using such a coherent anti-Stokes Raman scattering microscope, picosecond pulsed laser beams of narrow frequency bands (or, relatively broad pulse widths) need to be used. This is because that, when pulsed laser beams of broad frequency spectral bands are used, the frequency difference between these two pulsed laser beams may contain frequency difference components not corresponding to the specific molecular vibration frequency. These frequency difference components not corresponding to the specific molecular vibration frequency do not resonate with the specific molecular vibration frequency, and thus do not contribute to the generation of coherent anti-Stokes Raman scattering light. As a result, energies of two pulsed laser beams can not be efficiently utilized for the generation of coherent anti-Stokes Raman scattering light from the specific molecular vibration.
On the other hand, in the multiphoton excitation type laser microscope, for the purpose of increasing fluorescence excitation efficiency and of reducing the damage on the specimen to carry out the observation, femtosecond laser beams of broad frequency spectral bands (or, extremely narrow pulse widths) are used; in addition of which, these beams are in a state close to the Fourier-limited pulse. From the above reasons, the both observation methods with the coherent anti-Stokes Raman scattering microscope and the multiphoton excitation type laser microscope are different in the spec of pulsed laser beams to be used, and thus are difficult to achieve in a same microscope apparatus.